Ty3 is a retrovirus model element in S. cerevisiae that inserts into the transcription initiation site of genes transcribed by polymerase III. Many biochemical and genetic tools are now available with which to study Ty3. These include antibodies against Ty3 proteins, assays for nucleic acid intermediates, in vivo and in vitro assays for transposition, and a large group of mutants defective in Ty3 transposition. In the proposed work, aspects of the lifecycle which are less well understood, will be the focus, including integration site selection and effects of chromosomal context on targeting and expression of Ty3 in the chromosomal context, translocation into the nucleus, and the transition from viruslike particle to preintegration complex. There are five Specific Aims: 1) Identification of protein and DNA mediators of Ty3 position-specific integration. The minimal in vitro integration target for Ty3 is B' bound to the target gene. Recombinant reagents are available, including IN, RT, and NC with which to address the nature of the preintegration complex. The proteins that make direct contacts will be identified and the relative contributions of protein and DNA structures to target activity determined. 2) Understanding interactions between Ty3 and the chromosome. Ty3 is repressed by flanking pol III-transcribed genes and Ty3 appears not to access chromosomal 5S genes located in the rDNA repeat, although it readily uses plasmid-borne genes. The role of chromatin structure in these phenomena will be explored. The hypothesis that Ty3 acts as an insulator element for tRNA genes will be tested. 3) Identification of Ty3 and host components of the Ty3 nuclear translocation pathway. The Ty3 NLS was localized to the carboxyl-terminal region of IN in the current grant period and host mutants were identified which may affect Ty3 nuclear translocation. Experiments will address the composition of the Ty3 nuclear localized preintegration complex, whether this nucleoprotein complex uses the alpha-beta importin pathway used by consensus monopartite basic domain NLS-containing proteins, and whether subnuclear localization plays a role in Ty3 targeting. 4) Delineation of signals and mediators of the VLP to preintegration complex transition. The hypothesis that completion of reverse transcription is accompanied by specific modifications or rearrangements that signal uncoating resulting in nuclear entry will be tested. 5) identification of new host functions in the Ty3 lifecycle using genetic and biochemical approaches. The mechanism by which host mutations affect Ty3 tranposition will be examined for a set of fifty host mutants. The VLP-preintegration complex fraction will be analyzed for non- Ty3 proteins which are physically associated with Ty3 complexes and which may therefore affect the Ty3 lifecycle and the role of those proteins in the Ty3 lifecycle will he addressed.